Medical Biotechnology

Bernard R. Glick, Cheryl L. Patten, Terry L. Delovitch, Bernard R. Glick, Cheryl L. Patten, Terry L. Delovitch

• Publisher: ASM Press
• Year: 2013
• Print ISBN: 9781555817053
• eBook ISBN: 9781683673477
• Edition: 1
• Topic: Medicine
• Subtopic: Medical Microbiology & Parasitology

The future is now—this groundbreaking textbook illustrates how biotechnology has radically changed the way we think about health care

Biotechnology is delivering not only new products to diagnose, prevent, and treat human disease but entirely new approaches to a wide range of difficult biomedical challenges. Because of advances in biotechnology, hundreds of new therapeutic agents, diagnostic tests, and vaccines have been developed and are available in the marketplace.

In this jargon-free, easy-to-read textbook, the authors demystify the discipline of medical biotechnology and present a roadmap that provides a fundamental understanding of the wide-ranging approaches pursued by scientists to diagnose, prevent, and treat medical conditions.

Medical Biotechnology is written to educate premed and medical students, dental students, pharmacists, optometrists, nurses, nutritionists, genetic counselors, hospital administrators, and individuals who are stakeholders in the understanding and advancement of biotechnology and its impact on the practice of modern medicine.

Hardcover, 700 pages, full-color illustrations throughout, glossary, index.

SECTION I
The Biology behind the Technology

1. 1 Fundamental Technologies
2. 2 Fundamental Concepts in Immunology
3. 3 The Genetic Basis of Disease
4. 4 Immune Pathogenesis
5. 5 Microbial Pathogenesis

1
Fundamental Technologies

• Molecular Cloning
  • Preparation of DNA for Cloning
  • Insertion of Target DNA into a Plasmid Vector
  • Transformation and Selection of Cloned DNA in a Bacterial Host
  • Cloning Eukaryotic Genes
  • Recombinational Cloning
• Genomic Libraries
• Amplification of DNA Using PCR
• DNA Sequencing Technologies
  • Dideoxynucleotide Procedure
  • Pyrosequencing
  • Sequencing Using Reversible Chain Terminators
  • Sequencing by Ligation
• Sequencing Whole Genomes
  • Shotgun Cloning Strategy
  • High-Throughput Next-Generation Sequencing Strategies
• Genomics
  • Transcriptomics
  • Proteomics
  • Metabolomics
• SUMMARY
• REVIEW QUESTIONS
• REFERENCES

Molecular Cloning

Molecular biotechnology uses a variety of techniques for isolating genes and transferring them from one organism to another. At the root of these technologies is the ability to join a sequence of deoxyribonucleic acid (DNA) of interest to a vector that can then be introduced into a suitable host. This process is known as recombinant DNA technology or molecular cloning. A vast number of variations on this basic process has been devised. Development of the core technologies depended on an understanding of fundamental processes in molecular biology, bacterial genetics, and nucleic acid enzymology (Box 1.1). The beginning of the application of these technologies for the purpose of manipulating DNA has been credited to Stanley Cohen of Stanford University, Stanford, California, who was developing methods to transfer plasmids, small circular DNA molecules, into bacterial cells, and Herbert Boyer at the University of California at San Francisco, who was working with enzymes that cut DNA at specific nucleotide sequences. They hypothesized that Boyer’s enzymes could be used to insert a specific segment of DNA into a plasmid and then the recombinant plasmid could be introduced into a host bacterium using Cohen’s method. Within a few years, the method was used successfully to produce human insulin, which is used in the treatment of diabetes, in Escherichia coli. In the 25 years since the first commercial production of recombinant human insulin, more than 200 new drugs produced by recombinant DNA technology have been used to treat over 300 million people for diseases such as cancer, multiple sclerosis, cystic fibrosis, and cardiovascular disease and to provide protection against infectious diseases. Moreover, over 400 new drugs are in the process of being tested in human trials to treat a variety of serious human diseases.

Preparation of DNA for Cloning

In theory, DNA from any organism can be cloned. The target DNA may be obtained directly from genomic DNA, derived from messenger ribonucleic acid (mRNA), subcloned from previously cloned DNA, or synthesized in vitro. The target DNA may contain the complete coding sequence for a protein, a part of the protein coding sequence, a random fragment of genomic DNA, or a segment of DNA that contains regulatory elements that control expression of a gene. Prior to cloning, both the source DNA that contains the target sequence and the cloning vector must be cut into discrete fragments, predictably and reproducibly, so that they can be joined (ligated) together to form a stable molecule. Bacterial enzymes known as type II restriction endonucleases, or (more commonly) restriction enzymes, are used for this purpose. These enzymes recognize and cut DNA molecules at specific base pair sequences and are produced naturally by bacteria to cleave foreign DNA, such as that of infecting bacterial viruses (bacteriophage). A bacterium that produces a specific restriction endonuclease also has a corresponding system to modify the sequence recognized by the restriction endonuclease in its own DNA to protect it from being degraded.

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DNA

deoxyribonucleic acid

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mRNA

messenger ribonucleic acid

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box 1.1 The Development of Recombinant DNA Technology

Most important technologies are developed in small steps, and recombinant DNA technology is no exception. The ability to join DNA molecules from different sources to produce life-changing therapeutic agents like human insulin depends on the contributions of many researchers. The early 1970s were ripe for the development of recombinant DNA technology following the milestone discoveries of the structure of DNA by Watson and Crick (Watson and Crick, 1953) and the cracking of the genetic code by Nirenberg, Matthaei, and Jones (Nirenberg and Matthaei, 1961; Nirenberg et al., 1962). Building on this, rapid progress was made in understanding the structure of genes and the manner in which they are expressed. Isolating and preparing genes for cloning would not be possible without type II restriction endonucleases that cut DNA in a sequence-specific and highly reproducible manner (Kelly and Smith, 1970). Advancing the discovery by Herbert Boyer and colleagues (Hedgpeth et al., 1972), who showed that the RI restriction endonuclease from E. coli (now known as EcoRI) made a staggered cut at a specific nucleotide sequence in each strand of double-stranded DNA, Mertz and Davis (Mertz and Davis, 1972) reported that the complementary ends produced by EcoRI could be rejoined by DNA ligase in vitro.

Of course, joining of the restriction endonuclease-digested molecules required the discovery of DNA ligase (Gellert et al., 1968). In the meantime, Cohen and Chang (Cohen and Chang, 1973) had been experimenting with constructing plasmids by shearing large plasmids into smaller random pieces and introducing the mixture of pieces into the bacterium E. coli. One of the pieces was propagated. However, the randomness of plasmid fragmentation reduced the usefulness of the process. During a now-legendary lunchtime conversation at a scientific meeting in 1973, Cohen and Boyer reasoned that EcoRI could be used to splice a specific segment of DNA into a plasmid, and then the recombinant plasmid could be introduced into and maintained in E. coli (Cohen et al., 1973). Recombinant DNA technology was born. The potential of the technology was immediately evident to Cohen and others: “It may be possible to introduce in E. coli, genes specifying metabolic or synthetic functions such as photosynthesis, or antibiotic production indigenous to other biological classes.” The first commercial product produced using this technology was human insulin.

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A large number of restriction endonucleases from different bacteria is available to facilitate cloning. The sequence and length of the recognition site vary among the different enzymes and can be four or more nucleotide pairs. One example is the restriction endonuclease HindIII from the bacterium Haemophilus influenzae. HindIII is a homodimeric protein (made up of two identical polypeptides) that specifically recognizes and binds to the DNA sequence

(Fig. 1.1A). Note that the recognition sequence is a palindrome, that is, the sequence of nucleotides in each of the two strands of the binding site is identical when either i...

Table of contents

• Cover
• Table of Contents
• Preface
• About the Authors
• SECTION I: The Biology behind the Technology
• SECTION II: Production of Therapeutic Agents
• SECTION III: Diagnosing and Treating Human Disease
• Glossary
• Index
• End User License Agreement